Contributions of the Human Cytomegalovirus UL30-Associated Open Reading Frames to Infection.

Transposon-based insertional mutagenesis screens have assessed how disruption of numerous human cytomegalovirus (HCMV) open reading frames (ORFs) impacts in vitro viral replication. Insertional mutagenesis of the HCMV UL30 gene was previously found to substantially inhibit production of viral progeny. However, there are a number of putative UL30-associated ORFs, and it is unclear how they impact viral replication. Here, we report on the contributions of the eight UL30-associated ORFs to infection. We find that deletion of the canonically annotated UL30 ORF substantially reduces production of infectious virus at both high and low multiplicities of infection (MOI). This deletion likely has complex effects on viral replication, as we find that it reduces the expression of neighboring non-UL30-associated ORFs. Mutation of the initiating methionine of the canonical UL30 ORF indicated that it is dispensable for high- and low-MOI infection in the highly passaged AD169 strain, although it is important for low-MOI infection in the less-passaged TB40/E strain. Comutation of eight methionines in the UL30 region results in a low-MOI viral replication defect, as does mutation of the TATA box responsible for the most abundant UL30 transcript, which is found to be necessary for the accumulation of multiple UL30-associated protein isoforms during infection. In total, our data indicate the importance of the UL30-associated ORFs during low-MOI HCMV infection and further highlight the difficulty associated with the functional interrogation of broadly disruptive mutations: e.g., large deletions or transposon insertions.IMPORTANCE Viral genes and their products are the critical determinants of viral infection. Human cytomegalovirus (HCMV) encodes many gene products whose roles during viral infection have not been assessed. Elucidation of the contributions that various HCMV gene products make to infection provides insight into the infectious program, which could potentially be used to limit HCMV-associated morbidity, a major issue during congenital infection and in immunosuppressed populations. Here, we explored the role of HCMV's UL30-associated gene products and found that they are important for HCMV replication. Future work elucidating the mechanisms through which they contribute to viral infection could highlight novel avenues for therapeutic intervention.

The Human Cytomegalovirus UL38 protein drives mTOR-independent metabolic flux reprogramming by inhibiting TSC2.

Human Cytomegalovirus (HCMV) infection induces several metabolic activities that are essential for viral replication. Despite the important role that this metabolic modulation plays during infection, the viral mechanisms involved are largely unclear. We find that the HCMV UL38 protein is responsible for many aspects of HCMV-mediated metabolic activation, with UL38 being necessary and sufficient to drive glycolytic activation and induce the catabolism of specific amino acids. UL38's metabolic reprogramming role is dependent on its interaction with TSC2, a tumor suppressor that inhibits mTOR signaling. Further, shRNA-mediated knockdown of TSC2 recapitulates the metabolic phenotypes associated with UL38 expression. Notably, we find that in many cases the metabolic flux activation associated with UL38 expression is largely independent of mTOR activity, as broad spectrum mTOR inhibition does not impact UL38-mediated induction of glycolysis, glutamine consumption, or the secretion of proline or alanine. In contrast, the induction of metabolite concentrations observed with UL38 expression are largely dependent on active mTOR. Collectively, our results indicate that the HCMV UL38 protein induces a pro-viral metabolic environment via inhibition of TSC2.

Cofactor activity in factor VIIIa of the blood clotting pathway is stabilized by an interdomain bond between His281 and Ser524 formed in factor VIII.

The factor VIII (FVIII) crystal structure suggests a possible bonding interaction of His(281) (A1 domain) with Ser(524) (A2 domain), although the resolution of the structure (∼4 Å) does not firmly establish this bonding. To establish that side chains of these residues participate in an interdomain bond, we prepared and examined the functional properties of a residue swap variant (H281S/S524H) where His(281) and Ser(524) residues were exchanged with one another and a disulfide-bridged variant (H281C/S524C) where the two residues were replaced with Cys. The latter variant showed efficient disulfide bonding of the A1 and A2 domains. The swap variant showed WT-like FVIII and FVIIIa stability, which were markedly reduced for H281A and S524A variants in an earlier study. The disulfide-bridged variant showed ∼20% increased FVIII stability, and FVIIIa did not decay during the time course measured. This variant also yielded 35% increased thrombin peak values compared with WT in a plasma-based thrombin generation assay. Binding analyses of H281S-A1/A3C1C2 dimer with S524H-A2 subunit yielded a near WT-like affinity value, whereas combining the variant dimer or A2 subunit with the WT complement yielded ∼5- and ∼10-fold reductions, respectively, in affinity. Other functional properties including thrombin generation potential, FIXa binding affinity, Km for FX of FXase complexes, thrombin activation efficiency, and down-regulation by activated protein C showed similar results for the two variants compared with WT FVIII. These results indicate that the side chains of His(281) and Ser(524) are in close proximity and contribute to a bonding interaction in FVIII that is retained in FVIIIa.